Isolation of nucleic acids is an important step in molecular diagnostics. The quality and quantity of nucleic acids isolated from a sample greatly affects the success of downstream diagnostic applications. The clinical and field applications also demand that the isolation procedure be quick and amenable to automation.
Many procedures exist for isolating nucleic acids from various organisms and tissues. Some types of clinical and environmental samples present special challenges to successful isolation of nucleic acids. For example, certain tissues such as bone contain large amount of extracellular material that require removal before nucleic acids can be accessed. Some organisms, such as fungi, plants and bacteria possess cell walls or outer membranes that require harsh chemical treatment. The reagents used in the harsh treatments pose challenges for the downstream applications that utilize the isolated nucleic acids. Furthermore, degradation of the target nucleic acids during harsh treatment may lead to a false-negative result in the downstream detection assay. Yet to be clinically acceptable, a diagnostic procedure must have sufficient sensitivity, i.e. avoid false-negative results in patients' samples. Therefore in the field of molecular diagnostics, there is a need for improvement of the methods of isolating nucleic acids in order to make the diagnostic procedures sensitive, reliable and easy to perform.
A prerequisite for successful nucleic-acid based diagnostic test is isolation of undegraded, inhibitor-free nucleic acids. At the same time, there is a demand for simple, automation-friendly nucleic acid isolation procedures. Recently it has become popular to isolate nucleic acids using solid supports, such as for example, spherical microparticles. Especially popular are magnetic microparticles containing or coated with a glass-like substance, commonly referred to as “Magnetic Glass Particles” or “MGPs.” The nucleic acids isolation procedures employing MGPs require comparatively few steps and are easily automated. Especially popular are MGPs made by the sol-gel method, described in European publication EP 1 154 443 or U.S. Pat. Nos. 6,255,477 and 6,870,047.
The general descriptions and specific examples of MGPs made by the sol-gel method are readily available in the literature (see e.g. EP 1 154 443). These MGPs consist of a ferromagnetic core coated with silica-based glass-like material. The ferromagnetic core typically contains iron oxides, e.g. Fe3O4 or Fe2O3. The core may be a simple iron core, or may be made of a composite material. The core can also consist of a crystalline, ceramic or glass-like structure in which iron oxide is embedded. The glass coating may consist of amorphous material containing silicon oxide and further may contain one or more additional metal oxides such as boron oxide (B2O3), aluminum oxide (Al2O3), calcium oxide (CaO), barium oxide (BaO), potassium oxide (K2O), sodium oxide (Na2O), magnesium oxide (MgO) or lead oxide (Pb2O3). In some embodiments, the glass is silicon oxide and also contains one or more compounds in the following concentration range: B2O3 (0-30%), Al2O3 (0-20%), CaO (0-20%), BaO (0-10%), K2O (0-20%), Na2O (0-20%), MgO (0-18%), Pb2O3 (0-15%). The glass may also contain a smaller percentage (0-5%) of a number of other oxides such as Mn2O3, TiO2, As2O3, Fe2O3, CuO and CoO. Surfaces made of a composition of borosilicate glass have proven to be especially effective. Borosilicate glasses have a boron oxide content of more than 25%, e.g. a 70/30 composition of SiO2/B2O3.
The magnetic particles are sometimes modified with functional groups that facilitate the binding of nucleic acids. Such groups include, without limitation, poly-T oligonucleotides, for the capture of poly-A-containing nucleic acids, streptavidin, for the capture of biotin-labeled nucleic acids and specific probe sequences for the capture of nucleic acids containing the unique sequence complementary to the probe. However, most universally useful are magnetic particles with unmodified surfaces that are capable of isolating any nucleic acid present in the sample, regardless of the sequence.
A typical nucleic acid isolation protocol using MGPs commences with disruption of the cells or tissues in order to release the nucleic acids. The commonly used tissue disruption procedures are of chemical, enzymatic or physical nature, including ultrasound, high pressure, shear forces, strong bases, detergents or chaotropic agents, proteases or lipases. For chemical and enzymatic lysis, the lysis reagent typically includes a buffering agent, a salt, one or more of a denaturing substance and a chaotropic substance, a protease and optionally, a nuclease inhibitor and a preservative. The lysis reagent causes digestion of proteins, inhibition of nucleases, and solubilization of lipids, lipoproteins, and the like. For example, the buffering agent may be Tris, the salt may be a sodium, a potassium, an ammonium or a magnesium salt, such as a chloride or an acetate, the detergent may be sodium dodecyl sulfate, Triton-X or Tween, the chaotropic reagent may be urea, thio-urea, sodium iodite, sodium dodecyl sulfate, sodium perchlorate, guanidinium thiocyanate, guanidinium isothiocyanate or guanidinium hydrochlorite, the nuclease inhibitor may be a chelator such as EDTA, the preservative may be a metal azide, and the protease may be proteinase K. Typically, the sample is incubated with the lysis reagent at temperatures between 70 and 100° C., e.g., 90-95° C.
For most samples, the lysis reagents and conditions described above are sufficient to achieve the lysis of the cells and release nucleic acids into solution. Unfortunately, for some types of samples, lysis poses a major challenge. Some cells, organisms and tissues require harsh lysis conditions in order to break up the cell wall or tissue and release cellular contents. For example, Gram-positive pathogens such as Mycobacterium tuberculosis have lipid-rich peptidoglycan cell walls. There is a world-wide need for rapid methods of detecting M. tuberculosis and other mycobacteria. However, nucleic acid isolation is often a limiting factor for reaching desirable levels of assay sensitivity. See Neonakis et al. (2008) Molecular diagnostic tools in mycobacteriology, J. Microbiol. Methods, 75:111. Currently the desired sensitivity in a mycobacteria detection assay is achieved with a multi-step nucleic acid isolation procedure that includes repeated wash and centrifugation steps. See Shamputa et al. (2004) Molecular genetic methods for diagnosis and antibiotic resistance detection of mycobacteria from clinical specimens, APMIS, 122:728. Such procedure however is not automatable and not practical for most users.
In a typical method of isolating nucleic acids using MGPs, after the cellular compartments in the sample have been broken up to release the nucleic acids, the sample is brought into contact with MGPs in order to achieve binding of the nucleic acids to the MGPs. The MGPs may be added to the sample prior to lysis. For example, MGPs can be present in the vessel to which the initial sample is added. It has been found that the presence of MGPs does not affect lysis of the sample. Alternatively, the sample may be lysed first and MGPs introduced only after the lysis step is complete.
To achieve binding to MGPs, the sample is typically mixed with MGPs and incubated in this binding mixture for a period of time sufficient for the binding to occur. This step can be easily optimized by determining the quantity of immobilized nucleic acids on the surface of the magnetic glass particles at different points in time or by determining the yield of nucleic acids following different incubation times. Generally, incubation times of between 10 seconds and 30 minutes are appropriate for nucleic acids.
In most instances, the lysis reagent containing released nucleic acids is a suitable environment for the binding to MGPs to occur. However, in some instances, the lysis reagent makes the environment unsuitable for the binding of nucleic acids to the surface of magnetic glass particles. Especially where magnetic glass particles have unmodified surface, the binding between nucleic acids and the surface of the particles is dependent on conditions such as pH and ionic strength of the binding mixture. It has been found that maximum binding of nucleic acids to MGPs occurs at low pH, such as pH 5 or lower. However, for some applications, such low pH of the binding mixture may not be achieved. For example, the lysis reagent for lysing mycobacteria has a pH value of 12 or higher. In a typical procedure for isolating mycobacterial nucleic acids, the pH is lowered during a neutralization step following cell lysis but to not less than pH 9. At pH 9 or higher, the binding of nucleic acids to magnetic glass particles is inefficient. Up to the present time, this inefficiency of binding has been overcome by prolonged incubation times. This way of solving the problem is impractical for clinical applications. Furthermore, prolonged incubation threatens stability of the nucleic acid templates especially RNA templates.